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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 31, 2010 |
| Title |
brain-rep1 |
| Sample type |
SRA |
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| Source name |
whole brain
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| Organism |
Mus musculus |
| Characteristics |
genetic background: BALB-c
tissue: whole brain
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted from adult mouse tissues using trizol and PARE libraries prepared as described in German et al, Nature Protocols, 2008. Polyadenylated RNA was isolated from 200ug total RNA (from adult mouse brain, liver or lung) using oligo(dT) dynabeads (Invitrogen) and an RNA adaptor (5’-GUUCAGAGUUCUACAGUCCGAC-3’) ligated using T4 RNA ligase (Ambion). RNA was extracted with phenol / chloroform, ethanol precipitated and re-purified with oligo(dT) dynabeads. RNA was then reverse transcribed (SuperscriptIII, Invitrogen) using the primer (5’-CGAGCACAGAATTAATACGACT(18)V-3’) and amplified by PCR (Phusion DNA Polymerase, Finnzymes) using the primers (5’-GTTCAGAGTTCTACAGTCCGAC-3’ and 5’-CGAGCACAGAATTAATACGAC-3’). PCR conditions were 7 cycles of 94 °C for 30s, 60 °C for 20s and 72 °C for 3 min. Products were gel purified, cleaved with MmeI (New England Biolabs) and dephosphorylated (Shrimp alkaline phosphatase, New England Biolabs). Samples were run on a 12% polyacrylamide gel and a 42nt band excised. DNA was eluted from the gel overnight with 0.3M NaCl, filtered through a Millex 0.45uM column and ethanol precipitated. Products were then ligated using T4 DNA ligase (Ambion) to one of 6 double-stranded DNA adaptors (top 5’-P-TCGTATGCCGTCTTCTGCTTG-3’, bottom NN: 3’-NNAGCATACGGCAGAAGACGAAC-5’) that varied in the composition of an additional first 6 nucleotides (not in the given sequence) to enable barcoding of the separate tissue samples. Another 12% polyacrylamide gel was run and a 92nt band excised and purified as above followed by PCR amplification using the following conditions : 25 cycles of 94 °C for 20s, 60 °C for 20s and 72 °C for 20s. The product was again run on a polyacrylamide gel and purified prior to high-throughput sequencing using the Illumina GA platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
20nt from 5' monophospate RNA end
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| Data processing |
To generate the processed data BED files (with UCSC type track headers), we trimmed from the read ends up to tissue barcodes starts, leaving 20/21 nucleotides, removed all those with mitochondrial mappings (no-missmatch) and non-unique-genomic mapping (1missmatch permitted), removing also any with long homopolymeric tracts ( >9nt ) and low average read quality (< Phred 20 ). Allowing 1 missmatch, only mappings to unique locations in the mm9 genome were taken and non-unique read sequences consolidated into a single BED file entry having the read sequence as a name, followed by the accumulated number of identical reads. Read mapping was carried out with Bowtie using indexing against the UCSC mm9 genome sequences, the filtering and processing pipeline being Perl script based.
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| Submission date |
Jun 29, 2010 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Jan Marek Szubert |
| E-mail(s) |
marek.szubert@gmail.com
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| Organization name |
Centre for Cancer Biology
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| Lab |
Goodall
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| Street address |
Frome Rd
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| City |
Adelaide |
| State/province |
South Australia |
| ZIP/Postal code |
SA5000 |
| Country |
Australia |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE22627 |
PARE analysis of six mouse tissues to investigate cleavage products and degradome |
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| Relations |
| BioSample |
SAMN02196074 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM561025_mm9PARE1_BrainUni.bed.gz |
466.7 Kb |
(ftp)(http) |
BED |
| Processed data provided as supplementary file |
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